Absolute Configuration and Selective Trypanocidal Activity of Gaudichaudianic Acid Enantiomers

Gaudichaudianic acid, a prenylated chromene isolated from Piper gaudichaudianum, has been described as a potent trypanocidal compound against the Y-strain of Trypanosoma cruzi. We herein describe its isolation as a racemic mixture followed by enantiomeric resolution using chiral HPLC and determination of the absolute configuration of the enantiomers as (+)-S and (-)-R by means of a combination of electronic and vibrational circular dichroism using density functional theory calculations. Investigation of the EtOAc extract of the roots, stems, and leaves from both adult specimens and seedlings of P. gaudichaudianum revealed that gaudichaudianic acid is biosynthesized as a racemic mixture from the seedling stage onward. Moreover, gaudichaudianic acid was found exclusively in the roots of seedlings, while it is present in all organs of the adult plant. Trypanocidal assays indicated that the (+)-enantiomer was more active than its antipode. Interestingly, mixtures of enantiomers stowed a synergistic effect, with the racemic mixture being the most active.

Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi

Trypanosomes are flagellated protozoa responsible for serious parasitic diseases that have been classified by the World Health Organization as tropical sicknesses of major importance. One important drug target receiving considerable attention is the enzyme glyceraldehyde-3-phosphate dehydrogenase from the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (T. cruzi Glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH); EC 1.2.1.12). TcGAPDH is a key enzyme in the glycolytic pathway of T. cruzi and catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) coupled to the reduction of oxidized nicotinamide adenine dinucleotide, (NAD(+)) to NADH, the reduced form. Herein, we describe the cloning of the T. cruzi gene for TcGAPDH into the pET-28a(+) vector, its expression as a tagged protein in Escherichia coli, purification and kinetic characterization. The His(6)-tagged TcGAPDH was purified by affinity chromatography. Enzyme activity assays for the recombinant His(6)-TcGAPDH were carried out spectrophotometrically to determine the kinetic parameters. The apparent Michaelis-Menten constant (K(M)(app)) determined for D-glyceraldehyde-3-phosphate and NAD(+) were 352 +/- 21 and 272 +/- 25 mu M, respectively, which were consistent with the values for the untagged enzyme reported in the literature. We have demonstrated by the use of Isothermal Titration Calorimetry (ITC) that this vector modification resulted in activity preserved for a higher period. We also report here the use of response surface methodology (RSM) to determine the region of optimal conditions for enzyme activity. A quadratic model was developed by RSM to describe the enzyme activity in terms of pH and temperature as independent variables. According to the RMS contour plots and variance analysis, the maximum enzyme activity was at 29.1 degrees C and pH 8.6. Above 37 degrees C, the enzyme activity starts to fall, which may be related to previous reports that the quaternary structure begins a process of disassembly. (C) 2010 Elsevier Inc. All rights reserved.

Growth and antimicrobial activity of lactic acid bacteria from rumen fluid according to energy or nitrogen source

Objetivou-se avaliar os efeitos da suplementação com diferentes fontes de energia e de compostos nitrogenados sobre o crescimento e produção de bacteriocinas de bactérias ácido-láticas in vitro. As incubações foram conduzidas utilizando-se fluido ruminal originado de um novilho Holandês-Zebu fistulado no rúmen. O animal foi mantido em pastagem de Brachiaria decumbens recebendo 200 g/dia de proteína bruta suplementar. Os substratos e o inóculo foram acondicionados em frascos de vidro considerando-se oito tratamentos: celulose, celulose e caseína, celulose e peptona de soja, celulose e ureia, amido, amido e caseína, amido e peptona de soja e amido e ureia. Incubações sucessivas foram conduzidas para seleção dos microrganismos em função das fontes energéticas e de compostos nitrogenados. O amido favoreceu o crescimento de bactérias ácido-láticas em comparação à celulose. A suplementação com proteína verdadeira (peptona de soja e caseína) estimulou o crescimento dessas bactérias em comparação ao controle (sem suplementação com compostos nitrogenados). A adição de ureia não estimulou o crescimento de bactérias ácido-láticas. Nenhuma atividade antimicrobiana foi detectada a partir das colônias de bactérias ácido-láticas isoladas. Fontes de proteína verdadeira incrementam a competição entre bactérias fermentadoras de carboidratos estruturais e não-estruturais.

Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

Os autores padronizaram métodos para a avaliação da atividade da glicose-6-fosfato desidrogenase e glutationa redutase. O princípio geral do primeiro método baseou-se na formação de metahemoglobina pelo nitrito de sódio, seguido da estimulação da via das pentoses pelo azul de metileno. Foram estudados 46 indivíduos adultos, sendo 23 do sexo masculino e 23 do feminino, não deficientes em glicose-6-fosfato desidrogenase (G6PD), com idades variando entre 20 e 30 anos. Os resultados revelaram que a redução da metahemoglobina pelo azul de metileno para sangue total, foram de 154.50 e 139.90 mg/min (p<0.05) respectivamente para o sexo masculino e feminino. Para hemácias lavadas os valores foram de 221.10 e 207.85 mg/min (n.s.) respectivamente. Estas observações permitiram concluir que ao se empregar hemácias lavadas e 0.7 g% de concentração de nitrito de sódio, por um lado não houve diferença entre os sexos e por outro, abreviou o tempo de leitura da quantidade residual de metahemoglobina para 90 minutos. A avaliação da atividade da glutationa redutase foi feita baseado no fato de que a cistamina (agente tiol) liga-se aos grupos SH da hemoglobina formando complexos. Estes complexos são revertidos pela ação da glutationa redutase, ocorrendo conjuntamente nesta reação a redução da metahemoglobina. Foram estudados 32 indivíduos adultos, sendo 16 do sexo masculino e 16 do feminino, não deficientes em G6PD, com idades variando entre 20 e 30 anos. Os resultados revelaram valores de redução da metahemoglobina pela cistamina de 81.27 e 91.13 mg/min (p<0.01) respectivamente para o sexo masculino e feminino. Estas observações permitiram concluir que o emprego de hemácias lavadas e 0.1 molar de concentração de cistamina torna possível a leitura da quantidade residual de metahemoglobina aos 180 minutos de incubação. A atividade da glutationa redutase avaliada por meio da redução da metahemoglobina pela cistamina, foi estudada em 14 indivíduos do sexo feminino antes e após o tratamento com 10 mg por dia de riboflavina durante 8 dias. Os resultados foram de 73.69 e 94.26 mg/min (p<0.01) antes e após o tratamento. Estas observações permitiram concluir que a oferta de riboflavina, mesmo para indivíduos normais, aumenta a atividade da glutationa redutase. Foram ainda avaliados 3 indivíduos da raça negra e deficientes em G6PD, sendo 2 do sexo masculino e 1 do feminino. Houve ativação parcial da G6PD e glutationa redutase, sendo estas alterações mais intensas nos indivíduos do sexo masculino. Considerando-se a raça e as características laboratoriais observadas, foi possível sugerir que a deficiência em G6PD verificada é do tipo Africano, bem como, permitiu considerar os indivíduos do sexo feminino coin o sendo heterozigoto para esta deficiência. Por fim, a análise dos resultados em seu conjunto permitiu concluir que os métodos propostos se mostraram eficientes para avaliar a atividade da G6PD e glutationa redutase. Esta última é dependente da via das pentoses, geradora de NADPH e da riboflavina, vitamina precursora de FAD.

Osteoconductive Properties of beta-Tricalcium Phosphate Matrix, Polylactic and Polyglycolic Acid Gel, and Calcium Phosphate Cement in Bone Defects

Extensive bone defects in maxillofacial region can be corrected with autogenous grafts; otherwise, the disadvantages of the therapeutics modality take the research for new bone substitutes. The aim of the study was to evaluate and compare the osteoconductive properties of 3 commercial available biomaterials. A total of 30 calvarial defects (5-mm diameter) were randomly divided into 5 treatment groups, with a total of 6 defects per treatment group (n = 6). The treatment groups were as follows: 500 to 1000 Km beta-tricalcium phosphate (beta-TCP), polylactic and polyglycolic acid (PL/PG) gel, calcium phosphate cement, untreated control, and autograft control. The evaluations were based on histomorphometric analysis at 60 postoperative days. The results have shown that beta-TCP and autograft control supported bone formation at 60 postoperative days. beta-Tricalcium phosphate showed the highest amount of mineralized area per total area and statistically significant compared with PL/PG, calcium phosphate cement, and untreated control groups. The PL/PG gel does not have osteoconductive properties and performed similar to empty control. Calcium phosphate cement showed higher number of multinucleated giant cells around the sites of the biomaterial and showed newly formed bone only at the edges of the biomaterial, without bone formation within the biomaterial. The findings presented herein indicate that bone formation reached a maximum level when rat calvarial defects were filled with beta-TCP at 60 postoperative days. Further studies should be conducted with beta-TCP to understand the potential of this biomaterial in bone regeneration.

Intestinal anti-inflammatory activity of paepalantine, an isocoumarin isolated from the capitula of Paepalanthus bromelioides, in the trinitrobenzenesulphonic acid model of rat colitis

The preventative intestinal anti-inflammatory activity of paepalantine, an isocoumarin isolated from the capitula of Paepalarithus bromelioides, was tested in the trinitrobenzenesulphonic acid (TNBS) model of rat colitis. This was performed in two different experimental settings, i. e. when the colonic mucosa is intact or when the mucosa is in process of recovery after an initial insult. The results obtained revealed that the paepalantine pretreatment, at doses of 5 and 10 mg/kg, significantly attenuated the colonic damage induced by TNBS in both situations, as it was evidenced both histologically and biochemically. This beneficial effect was associated with an improvement in the colonic oxidative status, since paepalantine prevented the glutathione depletion that occurred as a consequence of the colonic inflammation. In addition, the intestinal anti-inflammatory effect exerted by this isocoumarin was associated with an inhibition of colonic nitric oxide activity, which is upregulated as a consequence of the inflammatory process. In conclusion, the preventative effect exerted by paepalantine in the TNBS model of rat colitis is probably related with its antioxidant properties.

Intestinal anti-inflammatory activity of esculetin and 4-methylesculetin in the trinitrobenzenesulphonic acid model of rat colitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Intestinal Anti-Inflammatory Activity of Baccharis dracunculifolia in the Trinitrobenzenesulphonic Acid Model of Rat Colitis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular approaches for structural characterization of Bothrops L-amino acid oxidases with antiprotozoal activity: cDNA cloning, comparative sequence analysis, and molecular modeling

Two L-amino acid oxidases (LAAOs) were identified by random sequencing of cDNA libraries from the venom glands of Bothrops moojeni (BmooLAAO) and Bothrops jararacussu (Bjussu LAAO). Phylogenetic analysis involving other SV-LAAOs showed sequence identities within the range 83-87% being closely related to those from Agkistrodon and Trimeresurus. Molecular modeling experiments indicated the FAD-binding, substrate-binding, and helical domains of Bmoo and Bjussu LAAOs. The RMS deviations obtained by the superposition of those domains and that from Calloselasma rhodostoma LAAO crystal structure confirm the high degree of structural similarity between these enzymes. Purified BjussuLAAO-I and BmooLAAO-I exhibited antiprotozoal activities which were demonstrated to be hydrogen-peroxide mediated. This is the first report on the isolation and identification of cDNAs encoding LAAOs from Bothrops venom. The findings here reported contribute to the overall structural elucidation of SV-LAAOs and will advance the understanding on their mode of action. (c) 2006 Elsevier B.V. All rights reserved.

Antioxidant activity, fatty acid profile and tocopherols of Tamarindus indica L. seeds

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Antimicrobial activity of an L-amino acid oxidase isolated from Bothrops leucurus snake venom

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Amino acid, Antioxidant and Ion Profiles of Carpolobia lutea Leaf (Polygalaceae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pectin methylesterase activity and ascorbic acid content from guava fruit, cv. Predilecta, in different phases of development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Indole ring oxidation by activated leukocytes prevents the production of hypochlorous acid

Hypochlorous acid (HOCl) released by activated leukocytes has been implicated in the tissue damage that characterizes chronic inflammatory diseases. In this investigation, 14 indole derivatives, including metabolites such as melatonin, tryptophan and indole-3-acetic acid, were screened for their ability to inhibit the generation of this endogenous oxidant by stimulated leukocytes. The release of HOCl was measured by the production of taurine-chloramine when the leukocytes (2 x 10(6) cells/mL) were incubated at 37ºC in 10 mM phosphate-buffered saline, pH 7.4, for 30 min with 5 mM taurine and stimulated with 100 nM phorbol-12-myristate acetate. Irrespective of the group substituted in the indole ring, all the compounds tested including indole, 2-methylindole, 3-methylindole, 2,3-dimethylindole, 2,5-dimethylindole, 2-phenylindole, 5-methoxyindole, 6-methoxyindole, 5-methoxy-2-methylindole, melatonin, tryptophan, indole-3-acetic acid, 5-methoxy-2-methyl-3-indole-acetic acid, and indomethacin (10 µM) inhibited the chlorinating activity of myeloperoxidase (MPO) in the 23-72% range. The compounds 3-methylindole and indole-3-acetic acid were chosen as representative of indole derivatives in a dose-response study using purified MPO. The IC50 obtained were 0.10 ± 0.03 and 5.0 ± 1.0 µM (N = 13), respectively. These compounds did not affect the peroxidation activity of MPO or the production of superoxide anion by stimulated leukocytes. By following the spectral change of MPO during the enzyme turnover, the inhibition of HOCl production can be explained on the basis of the accumulation of the redox form compound-II (MPO-II), which is an inactive chlorinating species. These results show that indole derivatives are effective and selective inhibitors of MPO-chlorinating activity.